Method and materials for producing deletion derivatives of polypeptides

ABSTRACT

The present invention describes an in vitro transposition-based methodology for generation of deletion derivatives of polypeptides. An artificial transposon containing at least partly within its transposon ends a modification with translation stop codons in three reading frames is provided. In the method, transposition complexes are assembled using the modified transposon and essentially random integrations into the target plasmid, containing a polypeptide coding nucleic acid of interest, are recovered as a plasmid pool. Subsequent manipulation steps including restriction enzyme digestions and ligation result in pools of mutant clones from which deletion derivatives of a polypeptide coding nucleic acid of interest and its respective deletion polypeptides could be produced.

The present invention relates to genetic engineering and especially invitro transposition. The invention describes a method and materials forproducing deletion derivatives of polypeptide coding nucleic acids. Inparticular, the invention provides means for efficient generation ofC-terminal deletions of polypeptides by the use of a modified transposonwith translation stop codons in all three reading frames. The inventionfurther provides a kit for producing said deletion derivatives.

BACKGROUND OF THE INVENTION

Thousands of different types of protein species constitute a majormolecular component of cellular life. These molecules are composed ofamino acid chains, the sequence of which is encoded by the genes in theorganism's DNA. The protein function can be diverse and specificfunctions have been evolved for different cellular demands. Native wildtype protein molecules can obviously be studied for their functionbiochemically and genetically. The data thus obtained can be informativebut very often such information is relatively limited. A betterdescription of protein function can be gained through mutationalanalysis in which various types of mutations are introduced into theprotein primary sequence and the mutated proteins are then analyzed fortheir function. With current recombinant DNA technology (Sambrook et al.1989, Sambrook and Russell 2001), generation of mutations is relativelyeasy and therefore mutational analysis of proteins has become a standardin functional studies of proteins.

In principle, three different types of mutations can be introduced intoa protein sequence (i) substitutions, (ii) insertions, and (iii)deletions. In a substitution mutation, a particular amino acid (or anamino acid stretch) in a protein is changed to another (or to anotheramino acid stretch of same length). In an insertion, an amino acid or astretch of amino acids is added to the protein thus increasing thelength of the amino acid chain. In a deletion mutation, an amino acid ora stretch of amino acids are eliminated from the protein sequence andthus the protein becomes smaller in size.

Various mutagenesis methods are currently available for generation ofdifferent types of mutations. These methods are typicallystraightforward to use. However, in most of the cases the wantedmutations are generated one by one and, therefore, their construction istime-consuming and labor-intensive. It would be desirable if a number ofmutations could be generated simultaneously. For certain types ofinsertion mutations this type of approach has been described (Hayes andHallet 2000). However, an efficient method for simultaneous generationof substitution and deletion mutations is still lacking.

One of the in vitro transposition systems we utilised for the presentinvention was a bacteriophage Mu-derived transposition system that hasrecently been introduced (Haapa et al. 1999a) and shown to functionefficiently in many types of molecular biology applications (Wei et al.1997, Taira et al. 1999, Haapa et al 1999ab, Vilen et al. 2001). Mutransposition proceeds within the context of protein-DNA complexes thatare called DNA transposition complexes or transpososomes (Mizuuchi 1991,Savilahti et al. 1995). These complexes are assembled from a tetramer ofMuA transposase protein and Mu-transposon-derived DNA-end-segments (i.e.transposon end sequences recognised by MuA) containing MuA bindingsites. When the complexes are formed they can react in divalent metalion-dependent manner with any target DNA and splice the Mu end segmentsinto the target (Savilahti et al 1995). In the simplest case, the MuAtransposase protein and a short 50 bp Mu right-end (R-end) fragment arethe only macromolecular components required for transpososome assembly(Savilahti et al. 1995, Savilahti and Mizuuchi 1996). Analogously, whentwo R-end sequences are located as inverted terminal repeats in a longerDNA molecule, transposition complexes form by synapsing the transposonends. Target DNA in Mu DNA transposition in vitro can be linear, opencircular, or supercoiled (Haapa et al. 1999a).

Mu transposition complex, the machinery within which the chemical stepsof transposition take place, is initially assembled from four moleculesof MuA transposase protein that first bind specific binding sites in thetransposon ends (FIGS. 5A and 5B). The 50 bp Mu right end DNA segmentcontains two of these binding sites (they are called R1 and R2 and eachof them is 22 bp long, Savilahti et al. 1995). When two ends, each boundby two MuA monomers, meet, the transposition complex is formed throughconformational changes, the nature of which are not fully understoodbecause of a lack of atomic resolution structural data on Mutranspososomes. However, the assembly of the minimal Mu transpososome isclearly dependent upon the correct binding of MuA transposase to Mu endsof the donor DNA. Thus, modifications in the conserved nucleotidesequence of transposon ends (e.g. R1 and R2 sequences in Mu R-end)should potentially have a negative effect on the efficiency of thetransposition since every altered nucleotide conceivably interferes withthe MuA binding. It has been documented (Lee and Harshey 2001, Coros andChaconas 2001) that the two last base pairs in the Mu transposon end canbe modified without severe effect on transpososome function. However, nodetailed analysis has been conducted for elucidation of the effects ofmodified R1 and R2 binding sites. In one example (Laurent et al. 2000) aNotI restriction site was engineered close to the transposon end thatchanged one base pair in the R1 sequence. In vivo studies indicate thatwithin the R1 and R2 sequences mutations generally have negative effectson transposition efficiency (Groenen et al. 1985, 1986). In addition,these effects are typically additive.

SUMMARY OF THE INVENTION

In this invention we describe a general methodology for making deletionderivatives of polypeptides using in vitro DNA transposition system. Themethod of the invention can be used to generate a number ofdeletion-derivatives of polypeptide coding nucleic acids simultaneouslyand with ease.

We utilised modified transposons that allowed us to generate C-terminaldeletion derivatives of polypeptides. The methodology should beapplicable to any protein, the encoding nucleic acid sequence (e.g. agene) of which is cloned in a plasmid or other DNA vector.

In one aspect, the invention features a transposon nucleic acidcomprising a genetically engineered translation stop signal in threereading frames at least partly within a transposon end sequence, orpreferably within transposon end binding sequence, recognised by atransposase.

In various embodiments the transposon nucleic acid of the invention maycontain a selectable marker and/or reporter gene. In one preferableembodiment the transposon end sequence of said transposon nucleic acidis Mu end sequence recognised by MuA transposase. In one particularembodiment said Mu end sequence is Mu R-end sequence.

In another preferred embodiment of the invention the modified transposonis a Tn7-derived transposon.

In a second aspect, the invention provides a method for producing adeletion derivative of a polypeptide coding nucleic acid comprising thesteps of:

-   (a) performing a transposition reaction in the presence of the    transposon nucleic acid of the invention and a target nucleic acid    containing a polypeptide coding nucleic acid of interest, (b)    recovering a target nucleic acid having said transposon nucleic acid    incorporated in said polypeptide coding nucleic acid.

In a preferred embodiment the method of the invention further comprisesa step of (c) expressing said polypeptide coding nucleic acid havingsaid transposon nucleic acid incorporated.

In a third aspect, the invention provides a kit for producing deletionderivatives of polypeptide coding nucleic acids. The kit comprises thetransposon nucleic acid of the invention.

In a fourth aspect, the invention features use of the transposon nucleicacid of the invention for producing deletion derivatives of polypeptidecoding nucleic acids.

The term “transposon”, as used herein, refers to a nucleic acid segment,which is recognised by a transposase or an integrase enzyme and which isessential component of a functional nucleic acid-protein complex capableof transposition (i.e. a transpososome).

The term “transposase” used herein refers to an enzyme, which is anessential component of a functional nucleic acid-protein complex capableof transposition and which is mediating transposition. The term“transposase” also refers to integrases from retrotransposons or ofretroviral origin.

The expression “transposition reaction” used herein refers to a reactionwherein a transposon inserts into a target nucleic acid. Essentialcomponents in a transposition reaction are a transposon and atransposase or an integrase enzyme or some other components needed toform a functional transposition complex. The method and materials of thepresent invention are exemplified by employing in vitro Mu transposition(Haapa et al. 1999ab and Savilahti et al. 1995) or transposition systemof Tn7 (Craig, 1996). Other transposition systems can be used as well.Examples of such systems are Tyl (Devine and Boeke, 1994, andInternational Patent Application WO 95/23875), Tn 10 and IS 10 (Kleckneret al. 1996), Mariner transposase (Lampe et al., 1996), Tc1 (Vos et al.,1996, 10(6), 755–61), Tn5 (Park et al., 1992), P element (Kaufman andRio, 1992) and Tn3 (Ichikawa and Ohtsubo, 1990), bacterial insertionsequences (Ohtsubo and Sekine, 1996), retroviruses (Varmus and Brown1989) and retrotransposon of yeast (Boeke, 1989).

The term “transposon end sequence” used herein refers to the conservednucleotide sequences at the distal ends of a transposon. The transposonend sequences are responsible for identifying the transposon fortransposition.

The term “transposon end binding sequence” used herein refers to theconserved nucleotide sequences within the transposon end sequencewhereto a transposase specifically binds when mediating transposition.

The term “target nucleic acid” used herein refers to a nucleic acidmolecule containing a protein coding nucleic acid of interest.

The term “translation stop signal” used herein refers to the geneticcode, which contains three codon triplets (UAA, UAG, UGA) forterminating the polypeptide chain production during protein synthesis ina ribosome. In a DNA strand the corresponding stop signal triplets areTAA, TAG and TGA.

The term “reading frame” used herein refers to any sequence of bases inDNA or RNA that codes for the synthesis of either a protein or acomponent polypeptide. The point of initiation of reading determines theframe, i.e. the way in which the bases will be grouped in triplets asrequired by the genetic code.

The term “genetic engineering” used herein refers to molecularmanipulation involving the construction of artificial recombinantnucleic acid molecules.

The term “gene” used herein refers to genomic DNA or RNA that aretranslated into polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.

Cat-Mu transposons: Cat-Mu containing wild type Mu ends, Cat-Mu(NotI)containing Mu ends with engineered NotI restriction site, which designis described in Laurent et al. 2000, and Cat-Mu(Stop×3) containing Muends with engineered translation stop signal in three reading frames(SEQ ID NO:2). Transposon end sequences (i.e. inverted terminal repeats)are drawn as rectangles.

FIG. 2.

Transposon end sequences of Cat-Mu transposons: Cat-Mu transposoncontaining wild type Mu ends (SEQ ID NO: 3 and 14), Cat-Mu(NotI)containing Mu ends with engineered NotI restriction site described inLaurent et al. 2000 (SEQ ID NO: 4 and 15), and Cat-Mu(Stop×3) containingMu ends with engineered translation stop signal in three reading frames(SEQ ID NO: 1 and 16) . Asteriks (*) show modified nucleotides in the Muends of Cat-Mu(NotI) and Cat-Mu(Stop×3).

FIG. 3.

Analysis of C-terminal deletion variants on DNA level. Plasmids bearingCat-Mu(Stop×3) transposon insertions (samples 1–24) were digested withBamHI, and they were analyzed on 1,8% agarose gels. The length of theshortest fragment of each digest corresponds roughly to the length ofthe deletion variant protein gene (0−˜650 bp). M=DNA standards.

FIG. 4.

Analysis of C-terminal deletion variants on protein level. The sizes ofthe deletion variant proteins, as predicted by sequencing analysis, aremarked below each lane as kilodaltons. M=molecular weight standard,C⁺=positive control, C⁻=negative control. Predicted deletion variantprotein products are pointed out by arrows.

FIGS. 5A and 5B

5A, Mu transposition complex. 5B, the assembly of Mu transpositioncomplex.

FIG. 6.

Overall strategy for production of C-terminal deletion variants of genesencoding proteins.

DETAILED DESCRIPTION OF THE INVENTION

It has been published previously that protein engineering applicationswill benefit from Mu-based transposon strategies since it wasestablished that any DNA sandwiched between Mu ends could be utilised asartificial transposons (Haapa et al. 1999a). In, principle insertionmutations (e.g. by addition of epitope tags or protein domains) anddeletion mutations (by addition of translation stop codons) wereforeseen with this strategy. However, introduction of a translation stopcodon between transposon ends would leave a number of encoded amino acidresidues into the protein's C-terminus. Given that an effective Mu endis about 50 bp in length, minimally this strategy would leaveapproximately 18 extra amino acids attached in the protein C-terminus.Extra amino acids may interfere with the protein function, therefore itwould be better to add the stop codons as close as possible to thetransposon end. By modifying the nucleotides of the Mu R-end (total of 7nucleotides were changed, 5 of said nucleotides reside in Mu R1sequence), we managed to place three stop codons in three reading framesvery close to the Mu R-end resulting in transposons that stillsurprisingly retained their ability to form transposition complexes thatwere competent for transposition chemistry, i.e. they facilitated theintegration of the transposon in vitro into a target plasmid. Inessence, all the possible C-terminal deletion variants can be generated.

We designed an artificial Cat-Mu(Stop)-transposon (SEQ ID NO:2)conferring resistance to chloramphenicol and Tn7-Kan(Stop)-transposon(SEQ ID NO:7) conferring resistance to kanamycin. Both contained intheir ends modified base pairs providing three stop codons in threereading frames (FIGS. 1 and 2). The gene mediating resistance tochloramphenicol is used as a selectable marker. The term “selectablemarker” refers to a gene that, when carried by a transposon, alters theability of a cell harboring the transposon to grow or survive in a givengrowth environment relative to a similar cell lacking the selectablemarker. The transposon nucleic acid of the invention preferably containsa positive selectable marker. A positive selectable marker, such as anantibiotic resistance, encodes a product that enables the host to growand survive in the presence of an agent, which otherwise would inhibitthe growth of the organism or kill it. The transposon nucleic acid ofthe invention may also contain a reporter gene, which can be any geneencoding a product whose expression is detectable and/or quantitatableby immunological, chemical, biochemical, biological or mechanicalassays. A reporter gene product may, for example, have one of thefollowing attributes: fluorescence (e.g., green fluorescent protein),enzymatic activity (e.g., luciferase, lacZ/β-galactosidase), toxicity(e.g., ricin) or an ability to be specifically bound by a secondmolecule (e.g., biotin). The use of markers and reporter genes inprokaryotic and eukaryotic cells is well-known in the art. In apreferred embodiment the transposon nucleic acid of the invention mayalso contain genetically engineered restriction enzyme sites. Forexample, the selectable marker gene within the transposon of theinvention may influence the protein expression when a construct obtainedby the method of the invention is inserted into a protein expressionplasmid. It is therefore desirable to engineer a pair of uniquerestriction sites to flank the selectable marker gene. The marker canthen be removed easily by the use of these sites and thus the finalexpression construct would not contain the marker gene.

Hence, one embodiment of the invention provides a transposon nucleicacid comprising a genetically engineered translation stop signal inthree reading frames at least partly within a transposon end sequence,or preferably within transposon end binding sequence, recognised by atransposase (i.e. at least one conserved nucleotide of the end sequencehas been modified, preferably two, three, four or more conservednucleotides have been modified). Preferably, the transposon nucleic acidof the invention comprises Mu or Tn7 transposon sequence. Morepreferably the transposon nucleic acid of the invention comprises MuR-end sequence, e.g., the sequence of SEQ ID NO:1 or SEQ ID NO:5 (Mu-Rend sequence not including 5′ overhang, which thus can vary). In atransposon end sequence of the transposon nucleic acid of the invention,translation stop signals of three reading frames are in 5′-to-3′direction, preferably in succession close to each other at a very end ofa transposon, thus the three stop signals are as near as possible theflanking sequence after the transposon is incorporated into a target.Furthermore, the transposon end sequences, which participate in theassembly of the transpososome discussed above, can be different fromeach other or they can be in different nucleic acid molecules.Preferably, both transposon end sequences participating in thetranspososome have similar sequences (i.e. they are located as invertedterminal repeats).

The transposon nucleic acid of the invention is exemplified here bytransposons of Mu (Examples 1–3) or Tn7 (Example 4) system. However, aperson skilled in the art understands that teachings of this inventioncan be utilised in other transposon systems as well.

Another embodiment of the invention is a method for producing a deletionderivative of a polypeptide coding nucleic acid comprising the steps of:

-   (a) performing a transposition reaction in the presence of a target    nucleic acid containing a polypeptide coding nucleic acid (e.g. a    gene) of interest and in the presence of a transposon containing a    genetically engineered translation stop signal sequence in three    reading frames at least partly within a transposon end sequence    recognised by a transposase, (b) recovering a target nucleic acid    having said transposon incorporated in said gene.

The transposition reaction (a) includes a transposon in a form of linearDNA molecule, transposase protein (e.g. MuA), and a target DNA asmacromolecular components. Additionally, the transposition reactioncontains suitable buffer components including Mg²⁺ ions critical forchemical catalysis. Buffer components such as glycerol and DMSO (orrelated chemicals or solvents) somewhat relax the requirements fortransposition reaction (Savilahti et al. 1995). Transposon DNA, inprinciple, can be of any length given that it in each end contain atransposon (e.g. Mu or Tn7) end sequence. Typically, target DNA is in aform of circular plasmid. However, any double-stranded DNA molecule morethan 25 bp is expected to serve as efficient target molecule (Savilahtiet al. 1995, Haapa-Paananen et al. 2002). In transposition reaction thereaction components are incubated together; during the incubationtransposition complexes first form and then react with target DNAsplicing the transposon DNA into target DNA. This process yieldstransposon integrations into target molecules. The stoichiometry of thereaction (excess target) generates target molecules each with a singleintegrated transposon. Most importantly, the integration site in eachmolecule can be different. Even though some sites in DNA are somewhatmore preferred than others most of the phosphodiester bonds in DNA willbe targeted (Haapa et al. 1999ab, Haapa-Paananen et al. 2002). Inpractice this means that the integration sites are selected essentiallyrandomly.

In the Examples below deletion mutant libraries were planned to coverthe gene of interest at least 10-fold, i.e. when the target gene wasapproximately 600 bp, the final pool should contain of a minimum of 6000mutants. As a test protein we utilised 23 kDa yeast Mso 1 protein (Aaltoet al. 1997). Those skilled in the art can easily design differentstrategies for mutant library construction as such strategies arewell-known in the art (see, e.g., Sambrook et al. 1989, Sambrook andRussell 2001).

A mutant library was produced as described in Example 2. Target nucleicacids with a transposon insertion were isolated by size-selectivepreparative agarose gel electrophoresis. A person skilled in the art maydesign different isolation methods as such methods are well-known in theart (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al, John Wiley & Sons: 1992). We screened individual deletionmutants by restriction analysis (FIG. 3). This analysis demonstratesthat in the library, there are variants of different sizes. A personskilled in the art can easily utilise different screening techniques.The screening step can be performed, e.g., by methods involving sequenceanalysis, nucleic acid hybridisation, primer extension or antibodybinding. These methods are well-known in the art (see, for example,Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley &Sons: 1992).

We sequenced 23 C-terminal mutants derived from Example 2. All themutants carried the translation stop codons in three reading frames.

Finally, the protein expression analysis (FIG. 4) demonstrated thatdifferent deletion variant proteins are produced. Probably due to lackof resolution in the utilised gel system, the supposedly expressedprotein was not detectable when the deletion derivative was 8 kDa orsmaller. Alternatively, very small versions of the Mso1 protein may beproteolytically degraded inside the cells.

A further embodiment of the invention is a kit providing means forproducing deletion derivatives of protein coding nuclear acid sequences.The kit comprises the transposon nucleic acid of the invention. The kitcan be packaged in a suitable container and preferably it containsinstructions for using the kit.

The results of the invention show that, unexpectedly, it is possible tosubstantially modify conserved sequences of transposon ends withoutcritically compromising the competence of the modified transposon toassemble transposition complexes and thereafter carry out transpositionchemistry. Thus, the invention provides a straightforward solution tothe problem of extra amino acids attached in the protein C-terminus ofthe deletion derivative which could be produced by a conventionaltransposition system, wherein the transposon used contains thetranslation stop signals between the transposon ends.

The present invention is further described in the following examples,which are not intended to limit the scope of the invention.

EXAMPLES Example 1

In Vitro Transposition Reaction

In vitro transposition reaction (25 μl) contained 720 ng cat-Mu(Stop)transposon as a donor, 500 ng plasmid pHis6-MSO1 as a target nucleicacid, 0.2 μg MuA, 25 mM Tris-HCl at pH 8.0, 100 μg/ml BSA, 15% (w/v)glycerol, 0.05% (w/v) Triton X-100, 126 mM NaCl and 10 mM MgCl₂. Thereaction was carried out at 30° C. for 4 h.

Further details and variables of in vitro Mu transposition are describedin Haapa et al. 1999ab and Savilahti et al. 1995, incorporated herein byreference.

Example 2

Generation of a Pool of Mutants with C-terminal Deletions in Mso1

In vitro transposition reactions with Stop-Mu were performed essentiallyas described in Haapa et al. (1999a) with the exception that theycontained 720 ng donor DNA (Stop-Mu×3) and 0,88 μg MuA. Ten reactionswere pooled, phenol and chlorophorm extracted, ethanol precipitated, andresuspended in 30 μl of water. Several 1 μl aliquots wereelectrotransformed, each into 25 μl of DH5α electrocompetent cells, asdescribed (Haapa et al. 1999a). Transposon-containing plasmid cloneswere selected on LB plates containing Ap and Cm. A total of ˜6×10⁵colonies were pooled and grown in selective LB-Ap-Cm medium at 37° C.for 3 h after which plasmid DNA was prepared from the pool with QiagenPlasmid Midi kit. This plasmid preparation was subjected to aXhoI-HindIII double digestion and preparative agarose gelelectrophoresis. The DNA fragment corresponding to transposon insertionsinto the Mso1-containing DNA fragment was isolated with QIAquick GelExtraction Kit (Qiagen). This fragment was then ligated into the plasmidpH is 6-MSO1 vector XhoI-HindIII backbone to generate a construct poolwith transposon insertions located only within the Mso1 gene. Afterligation, a pool of plasmids from ˜5×10⁴ colonies was prepared asdescribed above. Approximately 110 000 colonies were pooled.Transposon-carrying Mso1 fragments were cloned into clean vectorbackbone as described above and approximately 11 000 colonies werepooled in the final C-terminal deletion mutant library. At all stages,the transformants were selected with Ap and Cm.

Example 3

Restriction and Expression Analysis of Deletion Mutants

Mutant clones were analyzed for deletions by BamHI digestion and DNAsequencing. For protein expression analysis, single mutant plasmids wereintroduced into BL21(DE3) expression strain. Selective medium wasinoculated with o/n culture of bacteria containing mutant plasmid andgrown until OD₆₀₀ was 0,4–0,7. Protein expression was induced with 1 mMIPTG for 3 hours and samples were withdrawn for SDS-PAGE analysis.Bacterial lysates were run on 15% gels and stained with GelCode bluestain (Pierce) as recommended by the supplier.

Example 4

Generation of Deletion Mutants with Tn7-Kan (Stop) Transposon

In vitro Tn7 transposition reaction (20 μl) contained 40 ng Tn7-Kan(Stop) transposon (SEQ ID NO:7) as a donor, 100 ng plasmid pUC19 as atarget nucleic acid, 7 ng TnsA protein, 10 ng TnsB protein, 20 ng TnsC*protein, 25 mM Tris-HCl at pH 8.0, 50 μg/ml BSA, 2 mM DTT and 2 mM ATP.The reaction mixture was pre-incubated at 37° C. for 10 min beforeaddition of 30 mM magnesium acetate. After the addition the reaction wascarried out at 37° C. for 1 h.

The reaction mixture was precipitated with n-butanol to reduce the ionicstrength and to concentrate DNA prior to electroporation (Thomas, 1994)and resuspended in 10 μl of water. 5 μl aliquot was electrotransformedinto 50 μl of DH10B (Epicentre Technologies) electrocompetent cells.Transposon-containing plasmid clones were selected on LB platescontaining kanamycin (20 μg/ml). Approximately 20000 kanamycin resistantcolonies were recovered per 1 μg target DNA. Three clones were pickedfrom the transformation plates and grown in LB-Kn medium at 37° C.overnight after which plasmid DNA was prepared from the cultures withQiaPrep Spin Miniprep Kit. The Tn7-Kan (Stop) transposon insertion siteswere analyzed by DNA sequencing.

All the mutants carried the translation stop codons in six readingframes and in each case, the integrated transposon was flanked by a 5-bptarget site duplication generated in TnsABC*-mediated transposition.

MATERIALS AND METHODS

Bacteria, Media, Enzymes and Reagents

Bacterial cultures were grown in Luria broth supplemented withappropriate antibiotics: ampicillin (Ap) at 100 μg/ml, chloramphenicol(Cm) at 10 μg/ml and kanamycin (Kn) at 20 μg/ml when required.Escherichia coli strains were DH5α (Life Technologies), BL21(DE3)(Novagen), and DH10B (Epicentre Technologies). MuA protein was purifiedin collaboration with Finnzymes (Espoo, Finland) essentially asdescribed (Baker et al. 1993, Haapa et al. 1999a). TnsA, TnsB and TnsC*proteins were purchased from New England Biolabs. Restriction enzymesand T4 DNA ligase were from New England Biolabs and Promega, TritonX-100 from Roche. Standard DNA techniques were performed as described(Sambrook and Russell 2001). Enzymes were used as recommended bysuppliers. Sequencing was carried out at the sequencing service unit ofthe Institute of Biotechnology, University of Helsinki.

Plasmids and Transposons

Plasmid pHis6-MSO1 contains the 633 bp Mso1 gene as an insert (Aalto etal. 1997). The Cat-Mu(Stop) transposon (1254 bp) is a derivative of theCat-Mu transposon (Haapa et al. 1999a), and they encode resistance tochloramphenicol (FIGS. 1 and 2). The Cat-Mu(Stop)-transposon ends wereengineered to carry translation stop signals for both 5′-to-3′directions of dsDNA in all three reading frames. The Tn7-Kan (Stop)transposon is a derivative of the pGPS1.1 transposon (New EnglandBiolabs) and it encodes resistance to kanamycin. The Tn7-Kan (Stop)transposon ends were engineered to carry translation stop signals forboth 5′-to-3′ directions of dsDNA in all three reading frames. Tn7-Kan(Stop) transposon sequence is 4814 bp in length (SEQ ID NO:7) andnucleotides 3093–4791 set forth in SEQ ID NO:7 constitutes thetransposable element. Modified nucleotides were at the positions of3095, 3097, 3099, 3101, 3103, 4781, 4783, 4785, 4787, and 4789 set forthin SEQ ID NO:7.

Tn7-Kan (Stop) transposon was constructed from PCR-amplified fragments.The transposable fragment was amplified with primers 5′ acg gtg agt gagtag aaa ata gtt ggg aac tgg ga 3′ (SEQ ID NO:8) and 5′ cgt atg agt gagtag aat aaa gtc tta aac tga aca aaa tag a 3′ (SEQ ID NO:9) using theplasmid pGPS1.1 as template DNA (New England Biolabs) and the vectorfragment was amplified with primers 5′ aag tag ctt ttc tgt gac tgg t 3′(SEQ ID NO:10) and 5′ gat ggc atg aca gta aga gct 3′ (SEQ ID NO:11)using the plasmid pGPS1.1 (New England Biolabs) as template DNA.

Sequencing was performed using the primer 5 ′-gct agt tat tgc tca gcgg-3′ (SEQ ID NO:5). Sequencing of Tn7-Kan (Stop) transposon insertionsites in pUC19 plasmid was carried out using Model 4200 DNA Sequencer(LI-COR). Sequencing was performed using IRD700-labeled primers 5′ agctgg cga aag ggg gat gtg 3′ (SEQ ID NO:12) and 5′ tta tgc ttc cgg ctc gtatgt tgt gt 3′ (SEQ ID NO:13).

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1. A transposon nucleic acid having two transposon end sequences, atleast one of which comprises a genetically engineered translation stopsignal in three reading frames wherein one part of said translation stopsignal is within a transposon end binding sequence recognized by atransposase, and another part of said translation stop signal is betweensaid transposon end binding sequence and the distal end of saidtransposon end sequence.
 2. The transposon nucleic acid according toclaim 1, wherein said transposon nucleic acid contains a selectablemarker and/or a reporter gene.
 3. The transposon nucleic acid accordingto claim 1 or 2, wherein said one transposon end sequence is a Mu or Tn7end sequence.
 4. The transposon nucleic acid according to claim 3,wherein said transposon end binding sequence within said one Mutransposon end sequence is the Mu R-end binding sequence.
 5. Thetransposon nucleic acid according to claim 4, wherein said transposonsequence is set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:5.
 6. Thetransposon nucleic acid according to claim 3, wherein said transposonsequence is set forth in SEQ ID NO:7.
 7. The transposon nucleic acidaccording to claim 1, further comprising a genetically engineeredrestriction enzyme site.
 8. A method of producing a deletion derivativeof a polypeptide coding nucleic acid comprising the steps of: (a)performing a transposition reaction in the presence of a target nucleicacid containing a nucleic acid encoding a polypeptide of interest in thepresence of a transposon containing a transposon nucleic acid having twotransposon end sequences at least one of which comprises a geneticallyengineered translation stop signal in three reading frames wherein onepart of said translation stop signal is within a transposon end bindingsequence recognized by a transposase, and another part of saidtranslation stop signal is between said transposon end binding sequenceand the distal end of said transposon end sequences and (b) recoveringthe target nucleic acid having said transposon incorporated in saidpolypeptide-encoding nucleic acid.
 9. The method according to claim 8further comprising a step of (c) expressing said polypeptide-encodingnucleic acid having said transposon incorporated.
 10. The methodaccording to claim 8 or 9, wherein said transposon nucleic acid furthercomprises a selectable marker and/or a reporter gene.
 11. A kit forproducing deletion derivatives of polypeptide-encoding nucleic acidscomprising the transposon nucleic acid of claim 1.